Phorbol diester binding sites appear stable biologically, showing no significant variation in number, affinity for H-3-labeled phorbol-12,13-dibutyrate (PDBu) or down modulation in preneoplastic cells selected for resistance to 12-0-tetradecanoylphorbol-13-acetate (TPA)-induced mitogenesis or promotion. Nor does malignant transformation detectably perturb these binding sites in mouse or human cells. Loss of epidermal growth factor (EGF) receptors correlates with loss of mitogenic response to TPA in monolayer culture but has no effect on H-3-PDBu binding or TPA promotion of transformation. A partially purified preparation of transforming growth factor (TGF) from a human tumor line can promote anchorage-independent growth of a TPA-promotable mouse epidermal cell line lacking EGF receptors and responsiveness to EGF. I-125-labeled TGF shows specific binding to these EGF receptorless cells, suggesting that this TGF exerts its biological activity through receptors that are distinct from EGF receptors. The roles of membrane lipid methylation, reactive oxygen production and monovalent cation transport have been probed as possible mediators of signal transduction following receptor binding, and each has been found to be promotion relevant.